The effect of hyperthyroidism on cognitive function, neuroinflammation, and necroptosis in APP/PS1 mice

Background Increasing evidence has linked the thyroid dysfunction to the pathogenesis of dementia. Evidence from clinical studies has demonstrated that hypothyroidism is related to an increased risk of dementia. But the association of hyperthyroidism with dementia is largely unknown. Methods We used the adenovirus containing thyrotropin receptor (TSHR) amino acid residues 1-289 (Ad-TSHR289)-induced Graves’ disease (GD) phenotype in Alzheimer’s disease (AD) model mice (APP/PS1 mice) to evaluate the effect of hyperthyroidism on the cognitive function and β-amyloid (Aβ) accumulation. Results GD mice exhibited a stable long-term hyperthyroidism and cognitive deficits. Single Cell RNA-sequencing analysis indicated that microglia function played a critical role in the pathophysiological processes in GD mice. Neuroinflammation and polarization of microglia (M1/M2 phenotype) and activated receptor-interacting serine/threonine protein kinase 3 (RIPK3)/mixed lineage kinase domain–like pseudo-kinase (MLKL)-mediated necroptosis contributed to the pathological process, including Aβ deposition and neuronal loss. RIPK3 inhibitor could inhibit GD-mediated Aβ accumulation and neuronal loss. Conclusions Our findings reveal that GD hyperthyroidism aggravates cognitive deficits in AD mice and induces Aβ deposition and neuronal loss by inducing neuroinflammation and RIPK3/MLKL-mediated necroptosis. Supplementary Information The online version contains supplementary material available at 10.1186/s12967-023-04511-x.

of polyadenylated mRNA from single cells was performed within each GEM.After reverse transcription, the GEMs were washed, and the cDNA was amplified.The cDNA was fragmented, the fragment ends were repaired, and A-tails were added to the 3' end.The adaptors were ligated to fragments selected by double-sided solid-phase reversible immobilization (SPRI).Another double-sided SPRI selection was carried out after sample index PCR.The quality of the final library was assessed by checking the distribution of the fragment size using an Agilent 2100 bioanalyzer, and the library was quantified using real-time quantitative PCR (QPCR) (TaqMan Probe).The final products were sequenced using the Illumina HiSeq 4000 or Xten platform (BGI-Shenzhen, China).

Morris water maze (MWM)
The MWT test was performed as described previously.[1] The mice were habituated to the apparatus on day 0. Training trials were employed over 4 consecutive days (day 1-day 4), and the mice underwent the test three times a day.The location at which the mice were placed in the pool was changed in each trial, whereas the location of the hidden platform remained unchanged.On day 5 (probe trial), 24 hours after the previous training trial, the hidden platform was removed before conducting the tests.The cut-off time was set to 60 s.The percentage of time spent in the target quadrant and the time spent in the area where the hidden platform had been located were recorded to evaluate spatial memory capacity.

Novel object recognition test
The novel object recognition test is a behavioral test that is commonly used to investigate various aspects of mouse learning and cognition while being less stressful to the animals.[2] This test was performed over 3 days: the habituation day, training day, and testing day.On the habituation day, each mouse was placed in the middle of an open arena and allowed to freely explore for 10 min.On the training day, two identical objects were placed in opposite quadrants of the apparatus, and the mice were allowed to freely explore the objects for 10 min.On the testing day, one familiar object used on the training day and one novel object were placed in the same arena, and the mice were allowed to explore the objects.When both objects had been explored for 20 s or when 10 min had elapsed, the test was stopped.The discrimination index, which was calculated as the interaction time with the novel object divided by the interaction time with the novel object and the familiar object x 100, was calculated for each mouse.

Y maze test
The Y-maze test was to assess the short term spatial memory ability of the mice.[3,4] Each mouse was placed in one of the three arms and allowed to explore freely the maze for 10 min.An alternation was defined as successive entry into the three different arms on overlapping triplets.
Alternation behaviour (%) was calculated as the ratio of actual alternations to possible alternations (defined as the number of arm entries minus two).

Congo red staining
Congo red staining was used to determine whether tissue slices contained amyloid.[5] Brain sections (20 mm) were stained with Congo red working solution (NaCl-saturated 80% ethanol with 0.01% NaOH) for 10 min, incubated with solution B (Sigma-Aldrich, USA) for 35-40 min, dehydrated in 95% alcohol, incubated three times in xylene for 15 min, cleared in xylene and mounted with resinous mounting medium.Under a light microscope, amyloid deposits appeared red to orange.

Nissl staining
Nissl staining was used to visualize Nissl bodies in neurons.Staining was performed according to a previously described method.[4] The brain sections were rehydrated with distilled water before Nissl staining.After staining in 0.1% cresyl violet working solution (Sigma-Aldrich, USA) for 10 min, the sections were washed and then dehydrated with 95% ethyl alcohol for 8 s.The slices were dehydrated in 70% ethyl alcohol and 100% ethyl alcohol twice for 5 min each and cleared using xylene.Photographs of the brain sections were taken with a slide-scanning system (Tissue FAXS PLUS, Vienna, Austria).

Analysis of thyroid and liver function
The serum levels of total thyroxine (TT4) and thyrotropin (TSH) in the mice were quantitatively measured using enzyme-linked immunosorbent assay (ELISA) kit (Cusabio, Wuhan, China).The procedure of ELISA was conducted as described previously.[6] The sensitivities of the TT4 and TSH assays were 20 ng/ml and 0.15 μIU/ml, respectively, and the plate absorbance value was detected at 450 nm wavelength.
TSH receptor antibody (TRAb) and aspartate aminotransferase (AST) in the mice were quantitatively measured using ELISA kit (Jianglai Biotech, Shanghai, China) and a SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) according to the manufacturer's instructions.The sensitivities of the TRAb and AST assays were 0.2IU/L and 0.3 IU/L, respectively.

Tissue sample preparation
After the behavioural experiments (the 36 th week) were completed, brain tissue samples were collected.The mice were starved for 8 hours and then anaesthetized with isoflurane.Once anaesthetized, the mice were perfused with 0.9% NaCl.The hippocampus and cortex were separated to obtain samples for Western blotting.The samples were quickly stored in liquid nitrogen in a −80°C freezer before use.For immunofluorescence, the brains tissues were fixed by 4% paraformaldehyde in 4°C overnight followed by paraffin-embedding and serially sectioned at 20 µm thickness.

Western blotting
Western blotting was performed according to a previously described method.[7] Briefly, brain tissues were homogenized in ice-cold lysis buffer containing fresh protease and phosphatase inhibitor cocktail.After incubation for 30 min on ice, the lysates were centrifuged for 30 min (4°C, 12,000 × g), and the protein content in the supernatant was measured by Bicinchoninic Acid assay.

Immunoprecipitation
Immunoprecipitation is conducted as described previously.[8] Hippocampal tissues were lysed in lysis buffer containing phosphatase and protease inhibitors.The cell lysates were incubated with protein G agarose beads at 4°C for 4 hours.An MLKL antibody was added in lysis buffer for 4 hours at 4°C.The supernatants were immunoprecipitated with the antibody-coupled beads at 4°C overnight.The next day, the immunoprecipitated complexes were centrifugated and washed, andthe supernatant samples were subsequently subjected to routine western blotting analysis as described above.

Quantitative PCR (QPCR)
Quantitative PCR was performed using PrimeScript RT reagent Kit and SYBR Premix Ex Taq II (TaKaRa, Kusatsu, Japan) in a LightCycler 480 instrument (Roche).The 2 -ΔΔt method was used to analyze the targeted gene expression.All primers used for QPCR are shown in Supplementary Materials Table S1.

Thyroid histology
The formalin-fixed paraffin-embedded thyroid tissues were sectioned into 5-µm-thick sections, and fixed with tissue fixating solution for 15min.Subsequently, the sections were stained with Hematoxylin and Eosin (H&E) using standard experimental procedures.Microscope inspection, image acquisition and analysis were processed.

Enzyme linked immunosorbent assay (ELISA)
The levels of Aβ 40 , Aβ 42 (Thermo Fisher, Waltham, MA, USA), and β-secretase activity (Abcam, Cambridge, UK) in mouse hippocampal samples were analysed using ELISA kits according to the manufacturer's instructions.A SpectraMax microplate reader (Molecular Devices, Sunnyvale, CA, USA) was utilized to monitor the absorbance.

Primary microglial culture
Primary microglial cultures were performed as reported previously.[9] Briefly, the hippocampal tissues were removed from newborn APP/PS1 mice.The mixed glial cells were trypsinized and homogenized.The primary mixed glial cells were cultured for 16 days and isolated using a cell isolation kit (StemCell Technologies,Vancouver, British Columbia, Canada)according to the manufacturer's instructions.The oligomeric Aβ 1-42 peptide was prepared as described previously.[10] The primary microglia was treated with oligomeric Aβ 1-42 for 3 hours at a concentration of 5μmol/L.Supplementary

Fig
Fig S2 (A) Quantification of Aβ protein load in (2B).(B) Quantification of the NeuN-positive cell